Category A, B &C priority pathogens include biological agents that pose significant health threats and could be deployed as weapons of bioterror. The priority pathogens include a group of highly contagious RNA viruses that cause widespread illness and death in exposed population centers. This threat has galvanized the need for effective therapeutics that would inhibit production of infectious virions and resulted in PA-04-068, which calls for "development of assays for high-throughput drug screening". Ideal targets for therapeutic intervention are viral proteases that are produced inside cells of their human host and that function in the maturation of essential viral proteins. As such, high-throughput assays for rapid screening of large chemical and protein libraries are urgently needed to identify potent inhibitors of viral proteases. To meet this challenge, we developed a modular cell-based assay for pathogen proteases using the powerful genetic tools available in the model eukaryote, the yeast Saccharomyces cerevisiae. The modular nature of our assay permits development of protease assays for any pathogenic protease that can be expressed in the yeast cytoplasm. In the proposed studies, we will develop yeast assays for proteases produced by seven RNA viruses: Dengue Virus, West Nile Virus, Japanese Encephalitis Virus, Yellow Fever Virus, Hepatitis A Virus, SARS coronavirus, and Norwalk Virus. Specifically, we will engineer yeast strains that respond to the action of an intracellular protease by a cell-growth phenotype (Aims 1 &2). Our approach offers the genetic flexibility to select both for and against protease activity. We will then utilize a yeast assay already developed for the SARS 3C-like protease to conduct high-throughput compound screening for potent protease inhibitors (Aim 3). Through this study, we will establish an extensive series of high-throughput cell-based assays for proteases produced by category A, B &C biological agents and generate the first yeast-based screen for effective therapeutics against SARS coronavirus to counteract the threat produced by this highly virulent viral pathogen.